Journal ofMaterials Chemistry B

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aHistology Department, Iuliu Hatxieganu Un

Isaac Street 13, 400023 Cluj-Napoca, Rom

Fax: +40 264 592394; Tel: +40 264 406843bFaculty of Chemistry and Chemical Engine

Janos Street, RO 400028, Cluj-Napoca, Ro

ro; bianca@chem.ubbcluj.ro; Fax: +40 264cNational Institute for Research and De

Technologies, 65-103 Donath Street, RO

adriana.vulcu@itim-cj.ro; simina.dreve@iti

grosan@itim-cj.ro; liliana.olenic@itim-cj.ro;d“Ion Chiricutxa” Oncology Institute, 34-36

Romania. E-mail: mperde@iocn.ro; coratat

+40 264 598361eUniversity of Medicine and Pharmacy “Iuliu

Physiology, 1-3 Clinicilor Street, Cluj-Na

umfcluj.ro; Fax: +40-264-597257; Tel: +40-2fDepartment of Morphopathology, Universit

Medicine, 3-5 Calea Manastur Str., 40

pompeibolfa@gmail.com; Fax: +40 264 593gDepartments of Pharmaceutical Technology

University of Medicine and Pharmacy, 12

E-mail: machim@umfcluj.ro; Fax: +40 364hFaculty of Mathematics and Informatics,

Street, 1, 400023 Cluj-Napoca, Romania. E

264 591906; Tel: +40 264 405300

† Electronic supplementary informa10.1039/c3tb20476f

Cite this: DOI: 10.1039/c3tb20476f

Received 3rd April 2013Accepted 24th April 2013

DOI: 10.1039/c3tb20476f

www.rsc.org/MaterialsB

This journal is ª The Royal Society of

New nanomaterials for the improvement of psoriaticlesions†

Maria Crisan,a Luminita David,b Bianca Moldovan,b Adriana Vulcu,c Simina Dreve,c

Maria Perde-Schrepler,d Corina Tatomir,d Adriana Gabriela Filip,e Pompei Bolfa,f

Marcela Achim,g Ioana Chiorean,*h Irina Kacso,c Camelia Berghian Grosanc

and Liliana Olenic*c

The main purpose of the present paper is to emphasize the non-invasive effect of some new prepared

nanomaterials on skin diseases (psoriasis) together with the procedures to obtain them. These new

materials are based on gold nanoparticles and natural compounds extracted from native plants of the

Adoxaceae family (European cranberrybush – Viburnum opulus L. and European black elderberry –

Sambucus nigra L.) and possess a known anti-inflammatory activity mainly due to their high content of

anthocyanins and other polyphenols. The nanomaterials were characterized by transmission electron

microscopy (TEM), UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction

(XRD), energy-dispersive X-ray spectroscopy (EDX) and thermogravimetric analysis (TGA). Studies in vivo

and in vitro were made in order to determine the toxicity of the products. Based on the obtained

nanomaterials, specific dermatological creams were prepared. Their effect on psoriatic lesions, in

comparison with the hydrocortisone creams, was studied.

1 Introduction

It is well known that steroids (cortisone) used as topical treat-ment in psoriasis plaques give fast but temporary relief. They

iversity of Medicine and Pharmacy, Emil

ania. E-mail: maria.crisan@umfcluj.ro;

ering, Babesx-Bolyai University, 11 Arany

mania. E-mail: muntean@chem.ubbcluj.

590818; Tel: +40 264 593833

velopment of Isotopic and Molecular

400293, Cluj-Napoca, Romania. E-mail:

m-cj.ro; irina.kacso@itim-cj.ro; camelia.

Fax: +40 264 420042; Tel: +40 264 584037

Republicii Street, 400015 Cluj-Napoca,

@yahoo.com; Fax: +40 264 439260; Tel:

Hatieganu” Cluj Napoca, Department of

poca, Romania. E-mail: gabriela.lip@

64-598575

y of Agricultural Sciences and Veterinary

0372, Cluj-Napoca, Romania. E-mail:

792; Tel: +40 264 596384

and Biopharmaceutics, Iuliu Hatieganu

Ion Creanga St., Cluj-Napoca, Romania.

815170; Tel: +40 264 595770

Babesx-Bolyai University, Kogalniceanu

-mail: ioana@math.ubbcluj.ro; Fax: +40

tion (ESI) available. See DOI:

Chemistry 2013

are most useful for reducing inammation (redness, swelling,pain, pruritus), but in time tolerance occurs, and medicationbecomes less effective.

Also, long term usage of topical steroids results in atrophy,telangiectasia, etc. When used over large zones, a lot of corti-sone is absorbed into the body causing osteoporosis or immunesuppression.

A lot of work has been done to identify other anti-inam-matory products with similar effects but no side effects.

In this context, it has been discovered that plant originatedpolyphenolic compounds have potent antioxidant, anti-inammatory and immunomodulatory properties. The anti-inammatory effect of anthocyanins may be attributed to theprevention of the synthesis and release of pro-inammatorycompounds such as histamine, serine proteases, prosta-glandines, leukotrienes. As a result, polyphenolic compoundsoriginating from plants have been intensely studied in therecent years due to these remarkable properties.

In order to determine the most effective drugs with thesecharacteristics, researchers have discovered that metallicnanoparticles play an important role in drug delivery, generallyin biomedical science.1–3 The nanoparticles facilitate the pene-tration of the substances into the skin, enhancing their anti-mitotic, anti-inammatory and antimicrobial properties.4

Consequently, metallic nanoparticles have been prepared byusing a variety of methods.5–7

In recent years, many researchers have developed greenmethods for synthesizing nanoparticles using natural

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extracts.8–13 Along with the development of the concept of“green nanoparticles preparation”, initiated by Raveendranet al.,14 a large area of research has developed in this eld, dueto the fact that the preparation and use of non-toxic and eco-friendly nanomaterials is of great importance and has signi-cant applications in medicine.

Fruits from some Romanian native plants, part of theAdoxaceae family (European cranberrybush – Viburnum opulus L.and European black elderberry – Sambucus nigra L.) arefrequently used in folkmedicine for the treatment of menstrual,stomach and kidney cramps, duodenal ulcers, high bloodpressure, heart issues, asthma, arthritis, coughs and colds, evenconstipation.15–17

The biological activity of these berries is mainly due to theirhigh content of anthocyanins and other polyphenols.

In the present paper we describe new nanomaterials whichwere prepared by reducing the gold ions from HAuCl4 in thepresence of natural extracts (from European cranberrybush,denoted by E1, and European black elderberry, denoted by E2).The nanomaterials (AuNPs-E1 and AuNPs-E2) were character-ized from the morphological and structural point of view. Thenal products were tested in vivo and in vitro and the toxicityeffects were studied. From a medical point of view, our new bio-nanomaterials based on nanoparticles functionalized withnatural extracts were tested in comparison with cortisone inorder to assess their similar effects on psoriasis plaques whiledetecting no or fewer side effects. The medical results areconrmed by mathematical studies.

2 Experimental2.1 Materials and instruments

All chemicals and reagents used for extract preparation were ofanalytical grade, purchased from Merck Germany and usedwithout further purication. Tetrachloroauric acid waspurchased from Merck. All solutions were prepared in doubledistilled water.

The morphology and size distribution of nanoparticles wereexamined by a JEOL-JEM 1010 instrument (JEOL Inc). Theoptical absorption of synthesized nanomaterials was measuredwith a Shimadzu UV-Vis spectrometer (the wavelength rangebetween 300 and 850 nm at room temperature). The interactionof organic molecules with gold nanoparticles was investigatedby FT-IR spectroscopy. This was performed with a JASCO 6100spectrometer (spectral domain 5000–500 cm�1; the resolutionwas 4 cm�1 with the sample as KBr pellets). The crystallinenature of nanoparticles was assessed with a D8 AdvanceDiffractometer with CuKa1 radiation (l ¼ 15.4056 A) and Ge(111) monochromator. EDX analysis was performed with anOxford Instruments Analytical Limited, UK with INCA Energy300 soware. Thermogravimetric analysis (TGA) was performedusing a SDT Q600 (TA Instruments, USA) analyzer.

For in vitro cytotoxicity studies, normal keratinocytes(HaCaT) were purchased from the Cell Line Service of theGerman Cancer Research Center (Heidelberg, Germany). Todetermine IL1 and IL6 by ELISA, Quantikine kits from R&DSystems were used.

J. Mater. Chem. B

HaCaT cells were treated with nanoparticles and observed ona Zeiss AxioObserver D1 microscope equipped with a cooledAxioCamNR camera using AxioVision soware aer 24 hours ofexposure. Irradiation was performed in the culture plates(without lids) using a broadband UVB source Waldmann UV181. The substances absorbance was read on a Biotek Synergy 2microplate reader, at 492 nm and on a Tecan Sunrise reader.The corresponding concentrations were obtained based on thestandard absorption curve using the Magellan 3 soware.

Biochemical tests for evaluation of triglycerides, cholesteroland glucose were purchased from ChemiTech (France) andthose for glutamic oxaloacetic transaminase (GOT) and gluta-mic pyruvic transaminase (GPT) were obtained from Diag-nosticum Rt (Hungary). The hematological analyses (red bloodcell – RBC; hemoglobin concentration – Hb; hematocrit – Ht;white blood cell – WBC; platelets – PLT) were performed withSysmex XE 2100 Analyzer (Sysmex America, Inc.). All themicroscopic analyses were done using an Olympus microscope(BX41; Olympus). Pictures were acquired using a DP25 camera(Olympus) under a 400�magnication and processed using theCell B soware (Olympus).

For the assessment of psoriasis lesions, the non-invasive,high frequency ultrasound device Dermascan C 20 MHz (CortexTechnology, Denmark) was used.

2.2 Nanomaterials preparation

2.2.1 MethodsPlant materials. Natural extracts were obtained according to

Moldovan et al.18 To summarize, the frozen berry fruits werecrushed in a mortar. Five gram of fruits were transferred to anErlenmeyer ask and 200 mL double distilled water was added.The mixture was stirred for 1 h at room temperature and thenltered through Whatman no. 1 paper under vacuum. Theresulting plant material was re-extracted until a faint-colouredextract was obtained. The ltrates were quantitatively trans-ferred to a 250 mL volumetric ask and brought up to the markwith double distilled water. The total anthocyanin content fromcrude extracts was measured using a pH differential method.19

The major anthocyanin in E1 is cyanidin 3-O-glucoside andin E2, cyanidin 3-O-sambubioside. The ltrate solution was usedfor the synthesis of nanomaterials. The content of total antho-cyanins in E1 was calculated and turned out to be 11.32 mg l�1

and in E2, 111.80 mg l�1. This content was higher than the valuefound in other European cranberry fruits. The results indicatedthat the fruits of black elderberry represent a superior source ofanthocyanins, compared to those of the cranberry bush.

Synthesis of AuNPs-E1/E2 nanomaterials. The gold nano-particles were obtained by gold ions reduction with E1/E2 atpH ¼ 4. The pH of natural extracts solutions is modied at 7 forE1 and at 9 for E2 before adding to boiled HAuCl4 solution.

In order to obtain the gold nanomaterials (Fig. 1), we used aHAuCl4 (1%) solution and a solution of natural extract with aconcentration of total anthocyanins of 22.5 � 10�3 mM for E1,and 22.7 � 10�3 mM for E2 (aer dilution of natural extracts).As we stated before, the total anthocyanin content from crudeextracts was measured using a pH differential method.19

This journal is ª The Royal Society of Chemistry 2013

Fig. 1 TEM images of nanomaterials: (a) AuNPs-E1, (b) AuNPs-E2; (scale bar100 nm).

Fig. 2 UV-Vis spectra of nanomaterials: (1) AuNPs-E1 at 24 h after preparation;(2) AuNPs-E1 after 32 days from preparation; (3) AuNPs-E2 at 24 h after prepa-ration; (4) AuNPs-E2 after 32 days from preparation.

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The procedure to obtain the nanomaterials is as follows:12.5 ml natural extract were added to a vigorously stirred boilingsolution containing 150 ml bidistilled water and 5 ml HAuCl4.The solution was continuously stirred for 10 minutes. Gold ionreduction took place in 10 minutes in both cases and a red–purple colour was obtained for E1, and a red–mauve colour forE2 (Fig. S1c in ESI†). The colloids were stable for three weeks,aer which the nanomaterials aggregated very slightly and aer4 weeks they could not be used for the preparation of creams(Fig. 2).

Before being analyzed, the nanomaterials were centrifugedat 15 000 rpm for 10 minutes and washed and redissolved indouble distilled water. For XRD and FT-IR analyses they weredried at room temperature on watch glasses.

Aer nanomaterial centrifugation, the anthocyanin concen-tration in the remaining supernatant was determined. From thedifference between the anthocyanins concentration used atthe gold ions reduction and that found in the supernatant, theanthocyanin quantities bound on nanoparticles were obtained,which confers stability. The results of analytical experimentaldeterminations proved that from the initial quantity of anthocy-anin, 97.24% was bound on nanoparticles in the case of AuNPs-E1, and 99.6% in the case of AuNPs-E2 (unpublished work).

Calculated mass concentrations in gold were 149.20 ppm inboth cases and the corresponding calculated molar concentra-tions in gold nanoparticles are: AuNPs-E1 ¼ 0.08 nM andAuNPs-E2 ¼ 0.119 nM.

3 Results and discussions3.1 Nanomaterials analysis

3.1.1 Transmission Electron Microscopy (TEM) analysis.The study of nanomaterials by TEM (Fig. 1a and b) revealed that

This journal is ª The Royal Society of Chemistry 2013

gold nanoparticles were obtained of a size between 25 and90 nm for AuNPs-E1 and 11–70 nm for AuNPs-E2, with analmost spherical shape.

Nanomaterials obtained with E2 appear to be collected in theform of a bunch. For a period of 3 weeks, neither E1 nor E2extracts aggregated, as may be seen in Fig. S1a (in ESI†). FromTEM images, the average diameter of the nanoparticles wascalculated manually on 100 nanoparticles. The data is repre-sented by histograms of gold nanoparticles for each type ofnanomaterial (Fig. S1b, in ESI†).

3.1.2 UV-Vis spectroscopy. By UV-Vis spectroscopy weexamined the size and shape of the nanoparticles (Fig. 2). Aeraddition of the extract to the noble metal salt (when the boilingtemperature is achieved), the reaction began within minutesand aer 10 minutes a red–purple colour appeared, which wasclearly seen in the case of AuNPs-E1, and a red–mauve one inthe case of AuNPs-E2 (Fig. S1c, in ESI†).

The adsorption of organic molecules from natural extractson the surface of gold nanoparticles depends on the pH of theextract solution (Fig. S2, in ESI†). At pH 4 E1 and E2 are almostcolourless, so they have no absorption peaks in the 400–600 nmdomain. The surface plasmon resonance (SPR) band appearedat 543 nm for AuNPs-E1 and 546 nm for AuNPs-E2.

The gold nanoparticles were kept at refrigerator temperatureuntil used.

3.1.3 FT-IR spectroscopy. In the literature, there are manyFT-IR studies on nanomaterials obtained from naturalextracts.20–25 In the case of pure extracts (Fig. S3b and d, in ESI†)the stretching vibrations nOH can be observed which are char-acteristic of associated OH groups at 3405 cm�1 and 3381 cm�1

(nOH vibration band of sugar expected to appear around3400 cm�1). For nanomaterials, the spectra indicated that thevibrations were shied to higher values of 14 cm�1 for AuNPs-E1 and 28 cm�1 for AuNPs-E2. The band shis were greater inthe case of AuNPs-E2, which indicates that in this situation,more H linkages were broken.

The vibrations of aliphatic groups from sugar appearedunder an enveloped form at 2932 cm�1 (E1) and 2931 cm�1 (E2)while in the nanomaterials these vibrations were observed at2924 cm�1 and 2853 cm�1 (AuNPs-E1), and 2923 cm�1 and 2853cm�1 (AuNPs-E2).

Vibrations at 1732 cm�1 (E1), 1725 cm�1 (E2) were attributedto C]O from quinonoidal form (Fig. S2†). Aer the reduction ofAu3+ to Au0 with natural extracts, the peaks shied to 1709 cm�1

andmarkedly decreased in intensity. This is probably due to theadsorption of natural molecules from E1 and E2 at the surfaceof the gold nanoparticles, in a result similar to that obtained byKasthuri et al.20

The peaks at 1665 cm�1 (E1) and 1637 cm�1 (E2) are attrib-uted to C]C stretching vibrations. These peaks are shied tolower values in nanomaterials (1630 cm�1 in AuNPs-E1 and1628 cm�1 in AuNPs-E2).

Based on these data and the fact that the specic typicalpeaks assigned to the glycosidic units (1000–1200 cm�1) andnC–O from sugars around 1236 cm�1 appear in all cases, wesupposed that the anthocyanins were bound at the nanoparticlesurface.

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A possible mechanism for the formation of nanomaterialsbased on gold and natural E1 and E2 extracts is illustrated inScheme 1 for the majority of anthocyanins.

Anthocyanins from E1 and E2 present the structure (I) whichmay undergo reduction of Au3+ to Au0 yielding structure (II). Theanthocyanin carbonyl groups coordinate the gold reducedclusters and the molecule acts as ligand in order to prevent theagglomeration of particles. The FT-IR spectra agree with thisobservation.

3.1.4 XRD analysis. The diffraction peaks appeared at 2q ¼38.14�, 44.31�, 64.56�, 77.64�, 81.85� in intervals 2q between 5–85�, which are identical to those reported for standard goldmetal (Fig. S4 in ESI†). The peaks were assigned to the {111}facets of face-centered cubic metal gold structures. Theseobservations indicate that gold nanoparticles formed byreduction with E1 and E2 were dominated by the {111} facets.The XRD study clearly illustrates that AuNPs-E1/E2 materialsare crystalline in nature. The following dimensions wereobtained for crystals: DAuNPs-E1 ¼ 25 nm; DAuNPs-E2 ¼ 18 nm. It isimportant to note that nanoparticles dimensions observed inTEM images are higher than those obtained by XRD studiesbecause a nanoparticle may contain more crystallites.

3.1.5 EDX analysis. A large peak for gold metal and weakpeaks for oxygen and carbon obtained from organic moleculesbound to the surface of the gold nanoparticles can be noticed.The presence of Au0 is conrmed in the samples (Fig. S5 and S6,in ESI†).

3.1.6 TGA analysis. It is known that the decomposition ofanthocyanins is temperature sensitive. From the TGA prole, itcan be observed that the decomposition temperature of AuNPs-E1 shis towards higher values (800 �C) (Fig. S7 in ESI†). Thisindicates the stability of organicmolecules when they are boundto the gold surface. The shiing of the decomposition temper-ature towards higher values appears also in the case of aminoacid capped gold nanoparticles.26 Also, the mass percentage oforganic molecules bound to the gold clusters is approximately9.93% (we assumed that the rst step weight loss of 0.6746%represents desorption of water). The percentage is comparativeto the one obtained by EDX technique for AuNPs-E1: 12.15%.

The method we used in order to obtain the nanomaterialswas a “green” one, so no other synthetic chemicals were needed.

Scheme 1 Schematic diagram for AuNPs-E1/E2 formation.

J. Mater. Chem. B

The anthocyanin molecules from natural extracts play the roleof reduction compounds and at the same time are stabilisers ofgold nanoparticles, preventing their agglomeration.

In our study, the benecial effects of the new preparednanomaterials were emphasized aer their application in skindiseases.

3.2 Cytotoxicity and inammation studies

3.2.1 In vitro. The in vitro effects of the biomaterials wereevaluated on a spontaneously immortalized normal keratino-cytes cell line (HaCaT). Toxicity was assessed by microscopicobservations of alterations of cell morphology and evaluation ofviability by MTT test. The effects of the gold nanoparticles andextracts on the production of inammatory cytokines (IL1a andIL6) aer exposure to UVB (ultraviolet B radiation) were evalu-ated by ELISA.

Effect on cell morphology. HaCaT cells were treated withnanoparticles. The morphological changes as an effect of theexposure of HaCaT cells to AuNPs-E1/E2 were represented bygranulations in the cytoplasm, predominantly perinuclear,representing internalized nanoparticles (see Fig. 3b). Even athigher doses of AuNPs-E1/E2, no signicant alterations ofcell morphology occurred, except a higher amount of intra-cytoplasmatic granulation (Fig. 3c).

Effect on cellular viability. Cells were plated in 96-well plates,20 000 cells per well. Treatments were done aer 24 h with serialdilutions of each nanomaterial and extract solution, three wellsfor each concentration. At least three wells were le untreated(control).

Initial concentrations of the extracts (expressed in anthocy-anins) were 87 mg l�1 – E1 and 1108.6 mg l�1 – E2. Aer serialdilutions, the following concentrations were obtained: E1: 43.5;21.75; 8.7; 4.35; 2.175; 0.87; 0.435 mg l�1; E2: 554.3; 277.15;110.86; 55.43; 27.7; 11.08; 5.54 mg l�1. Aer 24 h incubationwith increasing concentrations of E1 and E2 extracts, theviability of HaCaT keratinocytes was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide].The tetrazolium salt (MTT) was reduced to a colouredcompound (formazan) only by metabolically active cells, byactivation of mitochondrial hydrogenase. Aer 24 h incubationwith increasing concentrations of extracts, the viabilitydecreased dose-dependently. For each extract, the concentra-tions which reduced viability with 50% (inhibitory concentra-tion 50 – IC50) were determined: IC50 E1 ¼ 3.992 mg l�1; IC50

E2 ¼ 79.4 mg l�1.

Fig. 3 Microscopic aspect of HaCaT cells without treatment (a) and treated withAuNPs-E1(15 mg ml�1) (b). Higher doses of AuNPs-E1 (150 mg ml�1) generatedmore intense intra-cytoplasmatic granulations (c). Magnification 200�.

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The AuNPs solutions had an initial concentration of 1.9 mgml�1. The concentrations used in the experiments were: 750;375; 150; 75; 37.5; 15; 7.5 mg ml�1; 24 h aer treatment, cellviability was assessed by MTT.

The viability of keratinocytes treated with Au nanoparticlesdecreased dose-dependently, the reduction being signicantonly at high concentrations (>37.5 mg ml�1) (Fig. 4). The AuNPsfunctionalized with E2 extract showed higher toxicity ascompared to the ones with E1 extract.

Effects on inammatory cytokines. HaCaT cells were seeded on24 well plates. Two plates were used: one was kept in the dark,while the other one was exposed to UVB (100mJ cm�2). The cellswere treated with 95 mg ml�1 nanoparticles (AuNPs-E1 andAuNPs-E2), 8.7 mg l�1 E1 extract and 110 mg l�1 E2 extract,30 minutes before exposure to UVB.

Supernatants were collected 24 and 48 h aer irradiation,and the inammatory cytokines (IL1a and IL6) were assessed byELISA, 24 and 48 h aer the treatment. IL1a levels increasedsignicantly aer HaCaT keratinocytes were exposed at 100 mJcm�2 UVB both aer 24 h and 48 h incubation. AuNPs-E1 andE2 extracts did not produce a signicant modication of IL1alevels in the supernatants of HaCaT cells when the cells werekept in the dark (Fig. 5a and b). When the cells were irradiatedaer the administration of AuNPs and extracts, 24 h later, the

Fig. 4 Cytotoxicity of AuNPs-E1 (-) and AuNPs-E2 (C) evaluated by MTT inHaCaT cells, 24 h after treatment. The viability of keratinocytes treated with goldNPs functionalized with extracts E1 and E2 decreased proportionally with NPconcentration, being significant only at high concentrations.

Fig. 5 IL1a concentration assessed by ELISA in the culture media of HaCaT cells24 h after UVB and treatment with AuNP E1 and E2 and extracts E1 and E2, (a);IL1a concentration 48 h after treatment with AuNPs and extracts (b); IL6concentration 24 h after UVB (c); IL6 after 48 h incubation (d). * – p < 0.05;** – p <0.01 and *** – p < 0.001.

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IL1a levels were comparable to the non-irradiated controls,while aer 48 h, the levels of this cytokine were signicantlyreduced in the cells pre-treated with AuNPs-E1 and AuNPs-E2.When the cells were treated with the extracts, the reduction wasless signicant (Fig. 5a and b).

IL6 levels increased signicantly aer exposure to UVB, bothaer 24 h ( p < 0.01) and 48 h (p < 0.001) (Fig. 5c and d). Goldnanoparticles functionalized with E1 and E2 extracts, and alsoE1 and E2 extracts, did not increase the levels of IL6 in thesupernatants of HaCaT cells. When the AuNPs and extracts wereadministered 30 min before exposure to UVB, IL6 levels werecomparable to those recorded in their non-irradiated counter-parts (Fig. 5c and d).

We can conclude that both the E1/E2 extracts and the goldnanoparticles functionalised with them showed an importantinhibitory effect on the secretion of pro inammatory cytokinesIL1a and IL6 aer irradiation with UVB. No signicant differ-ence between the reducing effects of the two types of AuNPs wasobserved.

3.2.2 In vivo. For in vivo studies 45 Female Wistar rats wereused. The animals were obtained from the Animal Departmentof “Iuliu Hatieganu” University of Medicine and Pharmacy,Cluj-Napoca, Romania.

All animal experiments were performed according to theOrganization for Economic Cooperation and Development(OECD) 434 guidelines (acute dermal toxicity-xed dose proce-dure) and were approved by the Ethical Committee on AnimalWelfare of the “Iuliu Hatieganu” University of Medicine inaccordance with the internationally accepted principles forlaboratory animal use and care (European Community Guide-lines, EEC Directive of 1986; 86/609/EEC). The animals wererandomly divided to 5 treatment groups of 9 animals each. Foracute dermal toxicity tests, two different concentrations ofAuNPs E1 and E2, 100 mg ml�1 and 1000 mg ml�1, were used.The control group was treated with PBS. All treated groupsreceived the above chemicals at 300 ml cm�2. At 1, 7 and 10 daysaer exposure skin biopsies were performed for histopatho-logical evaluations and blood was taken for biochemical(triglycerides, cholesterol, glucose, glutamic oxaloacetic trans-aminase – GOT and glutamic pyruvic transaminase GPT) andhematological investigations. The animals were maintainedand observed daily over 14 days for skin symptoms (edema,erythema, ulcers, bloody scabs, discoloration and scars) andtoxic signs (weight loss, water and food consumption,behavior). All animals were sacriced aer 14 days and the skinand liver were collected for routine histopathologicalexamination.

In acute dermal toxicity tests no deaths were recorded in the14 days of observation period in treated and control animals.No signicant changes in water and food consumption,behaviour and %weight gain of rats were observed for 14 days(data not shown). There were no signicant macroscopicchanges in the skin and fur of animals treated with nano-particles compared to the control group. Moreover, theappearance of the eyes and mucous membranes, respiratory,circulatory, nervous system and somatomotor activity wereunchanged.

J. Mater. Chem. B

Fig. 6 Skin and liver sections of Wistar rats following topical administration ofAuNPs-E1. No histological changes were visible at any time-point regardless of thematerial administered (HE; scale bar 200/100/200 mm).

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The hematological analysis in acute dermal toxicity testshowed no signicant changes in the levels of red blood cells(RBC), hemoglobin concentration (Hb), hematocrit (Ht), whiteblood cells (WBC) and platelets (PLT) in treated groupscompared to the control group. The biochemical parameters ofrats aer treatment with colloidal AuNPs-E1/E2 wereunchanged at all time intervals studied.

Following histopathological analysis we did not observe anytissue damage in the skin, nor in the liver of the rats, followingexposure to AuNPs-E1/E2 particles (see Fig. 6).

Fig. 7 Ultrasound images: (a) cutaneous histogram: normal aspect (b) psoriasisvulgaris: clinical aspect and histogram before treatment (c) psoriasis vulgaris:clinical aspects and histogram after treatment.

Fig. 8 Skin thickness (in mm) before and after treatment with: AuNPs-E1 cream(a – 8 patients); AuNPs-E2 cream (b – 7 patients); hydrocortisone cream for dermis(c–8 patients); hydrocortisone cream (d – 7 patients), for epidermis; moisturisingcream (e and f).

4 Application of new materials in psoriasis

Psoriasis vulgaris is a chronic inammatory disease character-ized by well-dened erythematous plaques bearing large,adherent, silvery scales, disseminated at cutaneous level. In thiscontext, we studied the anti-inammatory effect of our bioma-terials in the treatment of the skin lesions that appear inpsoriasis vulgaris. The study was performed on 8 subjects (forAuNPs-E1 and hydrocortisone creams) and 7 subjects (forAuNPs-E2 and also hydrocortisone creams), all aged between 35and 63, with clinical diagnosis of psoriasis. Patients enrolled inthe study signed an informed consent form. The study wasapproved by the Ethical Committee of the University of Medi-cine and Pharmacy “Iuliu Hatieganu” Cluj-Napoca, Romania.Pictures and sonograms (see Fig. 7) were acquired before andtwo weeks aer topically applied treatment (twice a day). Eachpatient used three preparations (a moisturising cream with andwithout active substances – AuNPs-E1/E2 and hydrocortisonecream). All subjects were submitted to an ultrasonographicevaluation, images from different areas of the skin being takenand analysed with the Dermavision soware. The ultrasoundevaluation allowed the acquirement of cross-sectional images ofthe skin up to a depth of 2.5 cm as well as the assessment of theechogenicity variation, by comparing the number of pixels (withdifferent echogenicity levels) before and aer therapy.

The clinical and ultrasonographic aspects were mathemati-cally assessed. The observation data were memorized in twovectors: the rst one with the values before treatment and thesecond one with the values aer the treatment. Computing thedistance between the two vectors (by means of the Euclideannorm), before and aer treatment with each cream, we provedthat the anti-inammatory properties of both studiedsubstances had similar effects on all subjects and were superiorto the effect of simple hydrocortisone cream. Moreover, theanti-inammatory effect was more intense for 3 patients who

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used the cream based on AuNPs-E1, but less intense for theothers (Fig. 8a). In contrast, this good effect may be observed forall patients who used the cream based on AuNPs-E2 (Fig. 8b).Concerning the hydrocortisone creams, the graphics in Fig. 8cand d also indicate an improvement of the psoriatic lesions, butnot as good as in the case of our bio-nanomaterial creams. InFig. 8e and f the experiment was performed with moisturisingcream. Aer daily topical application of the cream (3–4 times aday), a slight decrease of the epidermis can be noticed, whichcould be explained by the moisturizing effect of the cream,preventing scale accumulation.

At dermal level, a discrete increase of the thickness can benoticed, due to the humectant effect of the cream that preventsperspiration and ensures a profound hydrating effect. Thiseffect was observed and assessed by ultrasound, where anincrease of hypoechogenic pixels was noticed in the deepdermis.

These observations are sustained by the vector distancesbetween the data taken before and aer treatments.

5 Conclusions

Using an ecological, non-toxic method, we prepared two newnanomaterials based on gold nanoparticles and some naturalextracts. These newly prepared bio-nanomaterials can be usedsuccessfully in the medical eld, due to the biocompatibilityand stability offered by the organic compounds linked to metalnanoparticles.

Intercellular spaces between keratinocytes and pores of theskin, of micrometer order, allow the passage of the activesubstances from the epidermis into the dermis. Psoriaticlesions consist of thick layers of keratinocytes lled withscleroproteins. The nal goal of our research was to keep theactive substances (of nanometer order) on the epidermissurface for a long period of time, in order to modulate thesecretion of inammatory interleukins. This is the reason forendeavouring to maintain the large dispersions of the nano-particles diameters. In vitro and in vivo studies emphasized notoxicity of the nanomaterials with this large dispersion.Consequently, they are suitable for use in humans.

The obtained nanomaterials are viable for 3 weeks, if kept ata temperature of 4 �C. In vitro and in vivo studies showed a goodanti-inammatory effect which depends on the type of extractused. Comparing the used active substances, imaging andstatistical results, it was clear that the bio-nanomaterial AuNPs-E2 has the best anti-inammatory effect on psoriasis lesions,better than AuNPs-E1 and hydrocortisone. However, topicaltherapy in psoriasis should be personalized because it dependson the patient, the type of the lesions, their location, andpreviously used therapies, especially with hydrocortisone.

Acknowledgements

This work was elaborated in the frame project no. 147/2012,through the program “Partnerships in priority areas – PN II”,developed with the support of ANCS, CNDI – UEFISCDI.

This journal is ª The Royal Society of Chemistry 2013

Notes and references

1 G. Mie, Ann. Phys., 1908, 25, 377.2 M. C. Daniel and D. Astruc, Chem. Rev., 2004, 104, 293.3 Z. Zhong, S. Patskovskyy, P. Bouvrette, J. H. T. Luong andA. Gedanken, J. Phys. Chem. B, 2004, 108(13), 4046.

4 J. Lademann, H. Richter, A. Teichmann, N. Otberg,U. Blume-Peytavi, J. Luengo, B. Weiss, U. F. Schaefer,C. M. Lehr, R. Wepf and W. Sterry, Eur. J. Pharm.Biopharm., 2007, 66(2), 159.

5 J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot andA. Plech, J. Phys. Chem., 2006, 110, 15700.

6 M. Brust, J. Fink, D. Bethell, D. J. Schiffrin and C. Kiely,J. Chem Soc., Chem Commun., 1995, 1655.

7 L. Olenic, Gh. Mihailescu, S. Pruneanu, I. Bratu, G. Sigartau,L. B. Tudoran, Al. R. Biris and D. Lupu, Part. Sci. Technol.,2005, 23(1), 79.

8 V. Kumar and Y. S. Kumar, J. Chem. Technol. Biotechnol.,2009, 84, 151.

9 M. Amin, F. Anwar, M. R. S. A. Janjua, M. A. Iqbal andU. Rashid, Int. J. Mol. Sci., 2012, 13, 9923.

10 P. S. Vankar and D. Bajpai, Indian J. Biochem. Biophys., 2010,47, 157.

11 K. Badri Narayanan and N. Sakthivel, Mater. Lett., 2008, 62,4588.

12 S. S. Shankar, A. Ahmad, R. Pasricha and M. Sastry, J. Mater.Chem., 2003, 13, 1822.

13 S. P. Dubey, M. Lahtinen and M. Sillanpaa, Process Biochem.,2010, 45, 1065.

14 P. Raveendran, J. Fu and S. L. Wallen, J. Am. Chem. Soc., 2003,125, 13940.

15 C. Dennehy, J. Midwifery Womens Health, 2006, 51, 402.16 M. L. Altun, G. Saltan Citoglu, B. Sever Yilmaz and H. Ozbek,

Pharm. Biol., 2009, 47, 653.17 C. Kilham, Prepared Foods, 2001, 1, 39.18 B. Moldovan, O. Ghic, L. David and C. Chisxbora, Rev. Chim.,

2012, 63, 463.19 M. M. Giusti and R. E. Wrolstad, Current Protocols in Food

Analytical Chemistry, Unit F1.2, ed. R. E. Wrolstad, JohnWiley & Sons, New York, 2001, pp. 1–13.

20 J. Kasthuri, S. Veerapandian and N. Rajendiran, ColloidsSurf., B, 2009, 68, 55.

21 Z. Lin, W. Jianming, R. Xue and Y. Yang, Spectrochim. Acta,Part A, 2005, 61, 761.

22 J. Y. Song, H.-K. Jang and B. S. Kim, Process Biochem., 2009,44, 1133.

23 I. David, M. N. Stefanut, A. Cata, I. Ienascu, R. Pop,C. Tanasie and I. Balcu, J. Agroaliment. Processes Technol.,2009, 15(3), 348.

24 R. Reddy, C. Jayakumar, A. B. Morais, D. Sreenivasn andN. N. Gandhi, International Journal of Green Chemistry andBioprocess, 2012, 2, 1.

25 J. He, L. E. Rodriguez-Saona and M. M. Giusti, J. Agric. FoodChem., 2007, 55, 4443.

26 S. Aryal, B. K. C. Remant, N. Dharmaraj, N. Bhattarai,C. H. Kim and H. Y. Kim, J. Colloid Interface Sci., 2006,299, 191.

J. Mater. Chem. B